Bacterial production and refolding from inclusion bodies of a “Weak” toxin, a disulfide rich protein

The gene for the “weak” toxin of Naja kaouthia venom was expressed in Escherichia coli. “Weak” toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of “weak” toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia “weak” toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain “weak” toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of ¹²⁵I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α1₂β1_γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC₅₀ = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH₄C₁ cell line also showed the presence of activity close to that of the natural toxin (IC₅₀ 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain ¹⁵N-labeled analog of the neurotoxin.     

Reductive methylation as a probe of the heat-labile α-bungarotoxin-acetylcholine receptor membrane complex: Evidence for surface interactions

α-Bungarotoxin (α-Bgt) is a potent postsynaptic neurotoxin which blocks neurotransmission by binding very tightly to the acetylcholine-receptor (AcChR) protein. We have previously shown (P. Calvo-Fernandez, and M. Martinez-Carrion (1981) Arch. Biochem. Biophys., 208, 154–159) that α-Bgt free in its native solution conformation incorporates 12 methyl groups when reductively methylated using formaldehyde and sodium cyanoborohydride. We now show that when the α-Bgt molecule is bound to the AcChR contained in native membranes prepared from Torpedo californica electroplax, the number of accessible methylation sites is significantly reduced. This favors a model of α-Bgt-AcChR interaction involving significant numbers of lysyl moieties distributed over a reasonably large surface of the toxin molecule. In addition, this paper presents a novel procedure for the rapid and nondestructive dissociation of the toxin-AcChR membrane complex which takes advantage of the thermal instability of the complex.     

Assay of the acetylcholine receptor by affinity labeling

An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA), and for α-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophorus electricus and Torpedo californica and that this reaction is blocked in the presence of Naja naja siamensis α-neurotoxin and of other ligands of the acetylcholine binding site. Thus, in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct α-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 μg of receptor in the presence of 100-fold as much other protein.     

Reconstitution of High-Affinity Binding of a β-Scorpion Toxin to Neurotoxin Receptor Site 4 on Purified Sodium Channels

Abstract: Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1–3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a β-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). ¹²⁵I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min^?1 and 0.015 min^?1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with K_D of ～0.1 nM and a major class with a K_D of ～5 nM. Approximately 0.8 mol ¹²⁵I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1–3 or 5, consistent with the characteristics of binding of β-scorpion toxins to sodium channels in cells and membrane preparations. Our results show that specific, high-affinity binding at neurotoxin receptor site 4 on purified sodium channels can be restored by reconstitution into phospholipid vesicles and provide an experimental approach to analysis of the peptide components of the toxin receptor site.     

Functional studies with membrane-bound and detergent-solubilized α2-adrenergic receptors expressed in Sf9 cells

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery.We have expressed the three subtypes of α₂-adrenergic receptor (α₂-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of α_2B-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K_d values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that α₂-AR is active even in a lipid-free environment. The K_d values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of α₂-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.     

Inhibition of crotoxin binding to synaptosomes by a receptor-like protein from Crotalus durissus terrificus (the South American rattlesnake)

Crotoxin (Ctx) is a potent neurotoxin of the venom of Crotalus durissus terrificus (the South American rattlesnake). Ctx is a heterodimer composed of CB, a toxic PLA₂ subunit, and CA, a non-toxic and non-enzymatic subunit, that potentiates the neurotoxicity of CB in vivo. The deleterious action of Ctx upon C. d. terrificus snakes themselves is known to be prevented by a PLA₂ inhibitor (CNF) present in their blood serum. CNF acts by replacing CA in Ctx, thus forming a new stable complex CNF-CB. This complex no longer interacts with the target receptor (TR) to deliver CB to cause its lethal effect. Furthermore, CNF-CB seems to be reminiscent of the interaction Ctx-TR at the pre-synaptic site. In the present work, the binding competition between rat brain synaptosomes (TR) and CNF for Ctx was investigated. Radiolabeled Ctx, made of CA and one isoform of CB (CA-¹²⁵ICB₂), was used as ligand. The competition by unlabeled Ctx was taken as a reference. The potency of CNF as a competitor was evaluated under different incubation conditions with varying time scale addition of reagents (CA-¹²⁵ICB₂, synaptosomes and CA-CB₂ or CNF). CNF was able to inhibit the binding of the toxin to synaptosomes as well as to partially displace the toxin already bound to its membrane target. The mechanisms of competition involved were discussed and a previous schematic model of interactions between Ctx, TR and CNF was updated.     

Binding properties of Clostridium botulinum type C progenitor toxin to mucins

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327–333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl α2–3 lactose and sialyl α2–6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.     

Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors

A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.     

αB-Crystallin: A Hybrid Solid-State/Solution-State NMR Investigation Reveals Structural Aspects of the Heterogeneous Oligomer

Atomic-level structural information on αB-Crystallin (αB), a prominent member of the small heat-shock protein family, has been a challenge to obtain due its polydisperse oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on an ∼ 580-kDa human αB assembled from 175-residue 20-kDa subunits. An ∼ 100-residue α-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and C^β resonances have been obtained for residues 64-162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-crystallin domain have similar chemical shifts in both solid-state and solution-state, indicating similar structures for the domain in its isolated and oligomeric forms. Sites of intersubunit interaction are identified from chemical shift differences that cluster to specific regions of the α-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) ¹H-¹⁵N heteronuclear single quantum coherence spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer-monomer transition over the pH range 7.5-6.8. This steep pH-dependent switch may be important for αB to function optimally (e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia, which is accompanied by acidosis).     

A Large-Scale Expression in Escherichia coli of Neurotoxin II from Naja oxiana Fused with Thioredoxin

Neurotoxin II from the venom of cobra Naja oxiana is a short Type -neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [¹²⁵I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptor'sα, β, γ, orδ subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.     

Crystal Structure of the Antitoxin-Toxin Protein Complex RelB-RelE from Methanococcus jannaschii

Here we present the crystal structure of the Methanococcus jannaschii RelE-RelB (RelBE) toxin-antitoxin (TA) protein complex determined by the MIRAS (multiple isomorphous replacement with anomalous signal) method. The genes encoding this TA system are located in the chromosome of this archaeon and involved in stress response. RelE acts as an endoribonuclease that cleaves mRNA on the ribosome, and we compare the RelBE complex to the known structures of other TA systems belonging to this group and to endoribonucleases. M. jannaschii RelBE forms a heterotetramer with the antitoxin in the centre of the complex, a configuration that differs vastly from the heterotetramer structure of the previously published RelBE from another archaeon, Pyrococcus horikoshii. The long N-terminal α-helix of the tightly bound M. jannaschii antitoxin RelB covers the presumed active site of the toxin RelE that is formed by a central β-sheet, a loop on one side and a C-terminal α-helix on the other side. The active site of the M. jannaschii toxin RelE harbours positive charges that are thought to neutralize the negative charges of the substrate mRNA, including Arg62 that was changed to Ser62 by the Escherichia coli expression system, thereby leading to inactive toxin RelE. Comparative studies suggest that Asp43 and His79 are also involved in the activity of the toxin.     

Retarded protein folding of the human Z-type α1-antitrypsin variant is suppressed by Cpr2p

The human Z-type α₁-antitrypsin variant has a strong tendency to accumulate folding intermediates due to extremely slow protein folding within the endoplasmic reticulum (ER) of hepatocytes. Human α₁-antitrypsin has 17 peptidyl-prolyl bonds per molecule; thus, the effect of peptidyl-prolyl isomerases on Z-type α₁-antitrypsin protein folding was analyzed in this study. The protein level of Cpr2p, a yeast ER peptidyl-prolyl isomerase, increased more than two-fold in Z-type α₁-antitrypsin-expressing yeast cells compared to that in wild-type α₁-antitrypsin-expressing cells. When CPR2 was deleted from the yeast genome, the cytotoxicity of Z-type α₁-antitrypsin increased significantly. The interaction between Z-type α₁-antitrypsin and Cpr2p was confirmed by co-immunoprecipitation. In vitro folding assays showed that Cpr2p facilitated Z-type α₁-antitrypsin folding into the native state. Furthermore, Cpr2p overexpression significantly increased the extracellular secretion of Z-type α₁-antitrypsin. Our results indicate that ER peptidyl-prolyl isomerases may rescue Z-type α₁-antitrypsin molecules from retarded folding and eventually relieve clinical symptoms caused by this pathological α₁-antitrypsin.     

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal β-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.     

A modification of alpha-bungarotoxin amino group residues with retention of binding properties to isolated and membrane-bound acetylcholine receptor

α-Bungarotoxin (α-Bgt), an α-neurotoxin, has been ¹⁴C-methylated by treatment with [¹⁴C]formaldehyde following NaCNBH₃ reduction. The methylation rate is fast (about 84% methylation in 15 min), with 12 methyl groups incorporated per mole of α-Bgt or a mean of 1.7 methyl groups per available amine residue. The specific activity of α-[¹⁴C]Bgt is 768 mCi/mmol. Unlike most of the reported chemical modifications of α-neurotoxins, involving a high decrease of the toxin activity after modification, α-[¹⁴C]Bgt retains 100% of its unmodified ability to bind to both isolated acetylcholine receptor (AcChR) and AcChR-enriched membrane fragments prepared from Torpedo californica. This lysyl residue modification does not perturb the toxin binding activity, probably, because the net positive charges of the ?-amino groups and amino-terminal residue remain unaltered. ¹⁴C-Methylated α-Bgt appears better suited than ¹²⁵I-α-Bgt for use in AcChR binding studies because of the longer half-life of the isotope, and the apparent high uniformity of labeling of the toxin preparations.     

Crystal Structure of the HA3 Subcomponent of Clostridium botulinum Type C Progenitor Toxin

The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S-transferase and determined the three-dimensional structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 Å. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a-HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score > 10) were found. Especially, HA3a and HA3b domain I, mainly composed of β-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The electron density of liganded N-acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a-HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization,purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000–6000 pmol per mg protein of α-[¹²⁵]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius $\text{125 ± 10}$ Å and on sucrose gradient centrifugation one major peak was observed of 20–22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Identification and characterization of a taxon-specific three-finger toxin from the venom of the Green Vinesnake (Oxybelis fulgidus; family Colubridae)

Snake venoms contain a variety of protein and peptide toxins, and the three-finger toxins (3FTxs) are among the best characterized family of venom proteins. The compact nature and highly conserved molecular fold of 3FTxs, together with their abundance in many venoms, has contributed to their utility in structure-function studies. Although many target the nicotinic acetylcholine receptor of vertebrate skeletal muscle, often binding with nanomolar K_ds, several non-conventional 3FTxs show pronounced taxon-specific neurotoxic effects. Here we describe the purification and characterization of fulgimotoxin, a monomeric 3FTx from the venom of Oxybelis fulgidus, a neotropical rear-fanged snake. Fulgimotoxin retains the canonical 5 disulfides of the non-conventional 3FTxs and is highly neurotoxic to lizards; however, mice are unaffected, demonstrating that this toxin is taxon-specific in its effects. Analysis of structural features of fulgimotoxin and other colubrid venom 3FTxs indicate the presence of a “colubrid toxin motif” (CYTLY) and a second conserved segment (WAVK) found in Boiga and Oxybelis taxon-specific 3FTxs, both in loop II. Because specific residues in loop II conventional α-neurotoxic 3FTxs are intimately associated with receptor binding, we hypothesize that this loop, with its highly conserved substitutions, confers taxon-specific neurotoxicity. These findings underscore the importance of rear-fanged snake venoms for understanding the evolution of toxin molecules and demonstrate that even among well-characterized toxin families, novel structural and functional motifs may be found.     

Non conserved residues between Cqm1 and Aam1 mosquito α-glucosidases are critical for the capacity of Cqm1 to bind the Binary toxin from Lysinibacillus sphaericus

The Binary (Bin) toxin from the entomopathogenic bacterium Lysinibacillus sphaericus acts on larvae of the culicid Culex quinquefasciatus through its binding to Cqm1, a midgut-bound α-glucosidase. Specific binding by the BinB subunit to the Cqm1 receptor is essential for toxicity however the toxin is unable to bind to the Cqm1 ortholog from the refractory species Aedes aegypti (Aam1). Here, to investigate the molecular basis for the interaction between Cqm1 and BinB, recombinant Cqm1 and Aam1 were first expressed as soluble forms in Sf9 cells. The two proteins were found to display the same glycosilation patterns and BinB binding properties as the native α-glucosidases. Chimeric constructs were then generated through the exchange of reciprocal fragments between the corresponding cqm1 and aam1 cDNAs. Subsequent expression and binding experiments defined a Cqm1 segment encompassing residues S129 and A312 as critical for the interaction with BinB. Through site directed mutagenesis experiments, replacing specific sets of residues from Cqm1 with those of Aam1, the ₁₅₉GG₁₆₀ doublet was required for this interaction. Molecular modeling mapped these residues to an exposed loop within the Cqm1’s structure, compatible with a target site for BinB and providing a possible explanation for its lack of binding to Aam1.     

Single-channel study of the binding-gating coupling in the slowly desensitizing chimeric α7-5HT3A receptor

We have studied the role of the highly conserved residue αLysine145 in the early steps of activation by acetylcholine of the nicotinic acetylcholine receptor (nAChR). Both macroscopic and single-channel currents were recorded in the slowly desensitizing chimeric mutant receptor α7V201-5HT3A/R432Q/R436D/R440A, made of α7 nAChRs and serotonin receptors of subtype 3A (ch1), and its corresponding mutant K145A (ch1/K145A) expressed in Xenopus oocytes. Mutant ch1/K145A receptors had a reduced gating function similar to that produced by the same mutation in the wild type receptor α7. The mutated receptor has reduced opening rate constants, β, and increased closing rate constants, α.